Lightsheet Microscopy Overview Light sheet fluorescence microscopy (LSFM) is an imaging method that uses one or more illumination objectives to excite fluorophores in a specimen with a thin sheet of light. Emitted light from the sample is then visualized by a separate imaging objective that is perpendicular to the illuminating lightsheet. Light sheet microscopy (LSFM) has several advantages over confocal microscopy (LSCM): Excellent sample penetration- In conventional confocal microscopy, excitation illumination must pass from the objective through the sample to the plane of focus, then emitted light must return through the sample and into the objective via the same light path. The compounded scattering of both excitatory and emitted light limits sensitivity and resolution in thick samples. In LSFM, the excitation light sheet comes from dedicated illumination objectives and emitted light from the sample is detected by a separate imaging objective. This reduces light scattering and improves imaging in thick specimens. High speed- In confocal microscopy, the excitation laser scans across the sample field and emitted light is captured by a photo-multiplier tube, constructing an image pixel by pixel. In LSFM, The entire focal plane is illuminated at once, allowing an image to be collected using a high speed camera. Minimal phototoxicity and photobleaching- Because excitatory light is passing through the entire depth of the sample in confocal microscopy, there can be significant bleaching of the sample and a high risk for phototoxicity in live specimens. Because only a thin sheet of excitatory light is used in LSFM and because imaging with a camera is rapid, this method dramatically reduces photobleaching and phototoxicity. These properties make LSFM ideal for many biological imaging applications, including: Fluorescence imaging of thick samples Long term imaging of live samples For more information about LSFM, visit MicroscopyU. (Image courtesy of Carl Zeiss AG) Fluorescence Microscopy Comparison Chart Method Resolution Imaging Depth Speed Bleaching/Toxicity Wide-field good low (microns) fast low Confocal (LSCM)* ** good moderate (10s of microns) slower moderate/high Multi-photon** good good (100s of microns) slower moderate/high Light sheet (LSFM)* good good (100s of microns) fast low Super-resolution-SIM** very good low (microns) Super-resolution-STORM** excellent low (microns) Exact properties for each method are dependent on specific system configurations. General properties shown here are derived from published sources (Combs and Shroff, 2017). *Instruments available in A&S Imaging Center. **Instruments available in Light Microscopy Core. Comparison of Light Sheet Microscopes The A&S Imaging Center offers two microscopes capable of performing LSFM, the Zeiss Lightsheet Z.1 and the Leica SP8 DLS. They use very different implementations to create a light sheet (see individual microscope pages). The table below highlights operational differences between the two microscopes. Zeiss Lightsheet Z.1 Leica SP8 DLS Maximum sample width ~10mm ~4mm MInimum imaging volume ~20ml ~5ml Sample rotation ability Yes No Multiview acquisition Yes No Pivot scanning Yes No For more information or to discuss the best options for your imaging application, please contact us.
